Kinetic studies of allosteric catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa.

The pseudo-reverse reaction of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase in which arsenate is first coupled to citrulline followed by elimination of carbamylarsenate has been studied. Arsenate and citrulline saturation curves are sigmoidal. The different responsiveness of the transcarbamoylase to isosteric and allosteric ligands was examined both in the forward reaction, the carbamoylation of ornithine, and in the pseudo-reverse reaction, the arsenolytic cleavage of citrulline. Nucleoside monophosphates and polyamines that act as allosteric activators and inhibitors, respectively, on the carbamoylation reaction have the same effect on the rate of the arsenolytic cleavage of citrulline. ATP and other nucleoside triphosphates were found to stimulate enzyme activity at low carbamoylphosphate concentration with little influence on the carbamoylphosphate concentration at half-maximum velocity as well as on the cooperative index. When measuring the initial rate of the reverse reaction, the arsenolytic cleavage of citrulline, ATP was found to be a weak inhibitor, whereas CTP still stimulates the reaction and UTP was without influence. This unidirectional inhibition or activation phenomenon is likely apparent since initial studies were conducted and no consideration was given to equilibrium conditions. Regulation of catabolic OTCase by nucleoside triphosphates is without physiological meaning. In contrast, stimulation by nucleoside monophosphates may indicate that energy limitation could promote the synthesis and activity of the catabolic enzyme.

Proteinase inhibitor homologues as potassium channel blockers.

We report here the NMR structure of dendrotoxin I, a powerful potassium channel blocker from the venom of the African Elapidae snake Dendroaspis polylepis polylepis (black mamba), calculated from an experimentally-derived set of 719 geometric restraints. The backbone of the toxin superimposes on bovine pancreatic trypsin inhibitor (BPTI) with a root-mean-square deviation of < 1.7 A. The surface electrostatic potential calculated for dendrotoxin I and BPTI, reveal an important difference which might account for the differences in function of the two proteins. These proteins may provide examples of adaptation for specific and diverse biological functions while at the same time maintaining the overall three-dimensional structure of a common ancestor.

Sequence-specific 1H-NMR assignment and secondary structure of black mamba dendrotoxin I, a highly selective blocker of voltage-gated potassium channels.

The secondary structure of dendrotoxin I, an important constituent of the venom of the African black mamba snake Dendroaspis polylepis polylepis, was determined in aqueous solution by two-dimensional methods. Complete sequence-specific 1H-NMR assignment was obtained with the exception of the backbone amide proton of Gly39 and Cys40. Dendrotoxin I is based on a central antiparallel beta-sheet and two small helices located at the N- and the C-terminal extremities. These secondary-structural units occur at exactly the same places in the amino acid sequence as those of bovine pancreatic trypsin inhibitor (BPTI), with which dendrotoxin I shares 33% sequence similarity. According to the disulfide-bridge positions and the long-range NOE observed these secondary-structural elements fold in a similar manner to BPTI. This similarity allows an hypothesis according to which dendrotoxin I could derive from an ancestral Künitz-type proteinase inhibitor. This ancestor would have been heavily mutated at amino acid positions not critical for gross structure. The spatial locations of the solvent-exposed amino acids concerned could therefore serve as a guideline for interpretation of the structure/activity relationship of dendrotoxin I for the blockage of voltage-sensitive potassium channels of which dendrotoxin I is a strong inhibitor. The possible connections with other polypeptide toxins that block related ion currents is discussed.

Transport and phosphorylation of choline in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies.

When sycamore cells were suspended in basal medium containing choline, the latter was taken up by the cells very rapidly. A facilitated diffusion system appertained at low concentrations of choline and exhibited Michaelis-Menten kinetics. At higher choline concentrations simple diffusion appeared to be the principal mode of uptake. Addition of choline to the perfusate of compressed sycamore cells monitored by 31P NMR spectroscopy resulted in a dramatic accumulation of P-choline in the cytoplasmic compartment containing choline kinase and not in the vacuole. The total accumulation of P-choline over a 10-h period exhibited Michaelis-Menten kinetics. During this period, in the absence of Pi in the perfusion medium there was a marked depletion of glucose-6-P, and the cytoplasmic Pi resonance disappeared almost completely. When a threshold of cytoplasmic Pi was attained, the phosphorylation of choline was sustained by the continuous release of Pi from the vacuole although at a much lower rate. However, when 100 microM inorganic phosphate was present in the perfusion medium, externally added Pi was preferentially used to sustain P-choline synthesis. It is clear, therefore, that cytosolic choline kinase associated with a carrier-mediated transport system for choline uptake appeared as effective systems for continuously trapping cytoplasmic Pi including vacuolar Pi entering the cytoplasm.

Identification of inositol hexaphosphate in 31P-NMR spectra of Dictyostelium discoideum amoebae. Relevance to intracellular pH determination.

A sugar phosphomonoester, myo-inositol hexakisphosphate (phytic acid), has been identified as a major phosphorylated metabolite in Dictyostelium discoideum amoeba. Its intracellular concentration was estimated to be 0.7 mM. The identification was made in perchloric acid extracts on the basis of 31P-NMR chemical shift values and their variations with pH, by addition of authentic compound and by hydrolysis with wheat phytase. Perchloric acid extracts were prepared so as to avoid losses of insoluble salts of polyphosphorylated compounds with divalent cations. The glycolytic intermediate, 3-phosphoglycerate accumulated intracellularly in amoebae incubated in the presence of fluoride. The pH sensitive NMR signal of 3-phosphoglycerate was used to monitor cytosolic pH and a value of pH 7.4 was found in aerobic Dictyostelium amoebae.

In vivo 31P nuclear magnetic resonance studies of T1 and T2 relaxation times in rat brain and in rat brain tumors implanted to nude mice.

31P NMR spin-lattice (T1) and spin-spin (T2) relaxation times of phosphocreatine, ATP, inorganic phosphate, and phosphomonoesters have been measured in vivo at 4.7 T in rat brain and rat brain tumors implanted on nude mice. The relaxation data were acquired using a phase-cycled saturation-recovery spin-echo sequence. The problems associated with the phase modulation of the ATP lines by the homonuclear coupling constants were overcome by using selective refocusing pulses for the T2 measurements. In all the metabolites, large differences (1 to 2 orders of magnitude) are observed between the two relaxation times. T1 values in rat brain tumors are 30 to 90% longer than their counterparts in normal rat brain. T2 values follow the same trend with smaller variations except for phosphocreatine values which seem much less sensitive to the metabolic state of the tissues.

31P NMR measurements of T2 relaxation times of ATP with surface coils: suppression of J modulation.

The efficiency of selective refocusing pulses to suppress J modulation of spin echoes when using the surface-coil technique has been investigated. 31P T2 values obtained for ATP by the HAHN spin-echo method remain in good agreement when the standard refocusing pulse is replaced by selective pulses based on the DANTE excitation method.